A variety of methods for analyzing gene products and gene expression are available. Northern blot is one widely used method for monitoring gene expression (Sambrook et al). In this method, a cellular RNA fraction, typically total mRNA, is electrophoretically separated on a gel and the separated RNA species are transferred to a blot substrate. The RNA species are then hybridized with one or more labeled probes of interest, e.g., a labeled RNA or cDNA fragment on the substrate. The purpose of the method is to detect the size range and/or relative quantity of an expressed RNA species complementary to the labeled probe.
In general, sensitivity in the Northern blot is limited to mRNA species present at a level of about 1:10,000 of the total mRNA using radioactive detection, and Northern blots require up to 50 micrograms of mRNA per lane. Northern blots are labor intensive and in general not well suited to diagnostic applications.
The amplification step often required in Northern analysis may bias the relative abundance of discrete cDNA species in a complex mixture. Although methods which allow for reliable quantitation of the RT-PCR process (Piatak, et al) have been proposed, these are not practical for analysis of large numbers of genes.
It has also been proposed to analyze patterns of gene expression by hybridizing a gene of interest to colony blots of different cDNA libraries, where the frequency of hits provides a measure of “differential display” of the gene in the different tissues from which the cDNA libraries were originally made (Maser et al). This method requires the fabrication of a cDNA library and the labor-intensive analysis of unordered dot blots from the colony hybridizations.
Recently, there has been considerable interest in monitoring gene expression using tag sequencing. Here a cDNA library from a specific tissue type or disease state is made from poly A mRNA. Individual cDNA clones with inserts of 1-2 kb are selected at random and a “tag” of around 200 bases of each cDNA insert is sequenced. An expression profile is generated in the form of a computer database of the tag sequences for thousands of cDNA clones from the cDNA library (Orr). Computer analysis of expression profiles can determine which genes are differentially expressed in a specific tissue type or disease state. To date, however, the use of tag sequences has been limited by lack of information about the functional roles of most tag sequences, and in fact, assigning functional roles to tag sequences represents one of the challenging problems of the Human Genome Project.
In view of these limitations in identifying and quantitating gene expression levels for large numbers of expressed genes, it has not been practical heretofore to employ multi-gene expression as a sensitive test for cell status, e.g., in a disease state, or as a method for monitoring the effect of therapeutic treatment on diseased-state cells.